Haemophilus ducreyi is a Gram-negative bacterium that colonizes the genital epithelium and causes the sexually transmitted disease 'chancroid'. Despite its prevalence worldwide and its re-emergence in the United States, little is known about the molecular events leading to infection and disease that might lead to the development of better strategies to contain or eliminate this pathogen. In particular, how does this organism colonize genital epithelium, invade and multiply, and successfully evade host defenses? What is the chemical agent or agents responsible for causing genital ulcers? Based largely on structural and biological studies carried out in our laboratory several hypothesis can be proposed concerning the role of the outer-membrane lipooligosaccharides (or LOS) in these disease processes: Hypothesis 1. The LOS of H. ducreyi are major surface antigens and contain terminal sugars that are important in adhesion and invasion of human epithelial cells, and evasion of the human host defense response. Hypothesis 2. LOS is one of the principal cytotoxic agents responsible for skin lesions and ulcers. Hypothesis 3. A common structure or structural core exist in the LOS of all H. ducreyi. To examine these hypothesis, several specific aims are proposed to elaborate the role of LOS in chancroid: Specific Aim 1: Determine the structures of LOS from a diverse set of biologically important H. ducreyi strains. Specific attention will be given to determining the details of (i) the specific structural epitopes formed in the terminal oligosaccharide, (ii) the minimum conserved core region, i.e., X-Hepn-Kdo(P), (iii) the presence of sialic acid, and (iv) the structure and possible variation within the lipid A moiety. In addition, we will also identify the LOS glycoforms expressed under more biologically relevant conditions, such as LOS obtained from bacteria isolated from rabbit lesions and human cultured keratinocyte adhesion assays. Specific Aim 2: Assess the binding affinity of LOS to a series of vertebrate lectins using lectins previously isolated and characterized in Dr. Leffler's laboratory . Specific Aim 3: Determine the structure-cytotoxicity relationship of native and truncated LOS and lipid A. Specific Aim 4: Isolate and characterize specific enzymes involved in the biosynthesis of LOS, such as sialyltransferase(s) and CMP-NeuAc synthetase. Mass spectrometry techniques, including electrospray ionization mass spectometry, matrix-assisted laser desoprtion mass spectrometry and tandem MS/MS techniques will all be employed as the conerstone of these structural studies. Overall, structural data from LOS will be correlated to differences in biological function. This data should allow us to help unravel the molecular basis of pathogenesis of this organism. Obtaining such an understanding of LOS functions in these pathogenic processes will ultimately lead the way for the development of possible vaccines and other therapeutic strategies. For example, the presence of unique structural moieties in H. ducreyi LOS could be used to develop specific drugs acting as glycosyltransferase inhibitors. Alternatively, defining a common conserved LOS or LOS core structure among H. ducreyi strains could be exploited in developing a carbohydrate-based vaccine. It is our long-term goal to pioneer these efforts using this current proposed research effort to provide the necessary molecular details.